Factor VIII (FVIII) is mainly synthesized by hepatocytes and sinusoidal endothelial cells. The plasma concentration of FVIII is comprised between 0.1 and 0.2 mg/l; the circulating form is inactive and associates with von Willebrand factor (vWF). FVIII plays a key role in the endogenous (so-called intrinsic) pathway of blood coagulation. When a blood vessel is damaged by trauma, bleeding is triggered. In response, the process of hemostasis is initiated, consisting of a complex chain of events leading to the formation of a blood clot which seals the site of injury. Blood coagulation begins when platelets adhere to injured vessel walls. If the injury is severe, the platelet aggregates at the site of injury are insufficient to form a hemostatic plug to staunch the blood flow. Thus coagulation factors intervene whose purpose is to form the fibrin network, generated from soluble fibrinogen molecules by the action of thrombin. The formation of this network composed of insoluble fibers is crucial to firmly anchor the blood clot. Cascade shall be understood to mean that, sequentially and at each step, a precursor protein is converted to an activated protease which cleaves or acts as cofactor for cleavage of the next precursor protein of the cascade. Thus, FVIII is proteolytically cleaved in FVIIIa by the action of thrombin and factor Xa. In this active procoagulant form (FVIIIa), FVIII strikingly increases the proteolytic efficiency of factor FIXa towards factor FX.
Hemophilia A is a bleeding disorder characterized by a deficiency of activated FVIII due to a mutation in the recessive gene encoding FVIII. In some rare cases, hemophilia A may arise from the spontaneous development of auto-antibodies directed against FVIII; this is known as acquired hemophilia A.
Hemophilia is manifested as a defect of blood clotting in response to a hemorrhage. Untreated type A hemophiliacs exhibit symptoms such as excessive bleeding after trauma and sometimes even spontaneous hemorrhages, particularly into the articulation joints. Hemophilia A is the most common coagulation disorders and occurs in 1 in 5,000-10,000 male births. Not all hemophiliacs are affected in the same manner or to the same extent. For instance, hemophilia A is considered i) severe when FVIII levels are less than or equal to 1% of “normal” circulating levels; ii) moderate when FVIII levels are within the range of 1 to 5% of “normal”; and iii) mild when FVIII levels are between 5 and 30% of normal. These three types of hemophilia A occur at the following frequencies: 50% of hemophiliac patients have the severe form, 10% the moderate form and 40% the mild form.
Many genetic abnormalities have been associated with the gene coding for FVIII. Said gene is located at the tip of the long arm of the X chromosome (locus Xq28). Hemophilia A results from an abnormality in this gene. It is an X-linked recessive disorder: males and females can transmit the disorder but only males are affected. The molecular defects may be gene mutations, deletions or inversions. The majority of patients harboring missense point mutations have mild or moderate disease. Deletions are classified into two types: i) small deletions; ii) large deletions (>1 kb). Most large deletions confer a severe phenotype. With respect to genetic inversions, the intron 22 inversion is the most frequent and is responsible for the majority of cases of severe hemophilia A (45%). Another inversion, the intron 1 one, can cause severe disease while less frequent (3%).
In summary, these mutations result in either a decreased production of functionally normal FVIII molecules, or a quantitatively normal production of functionally defective FVIII molecules.
The FVIII gene codes for a polypeptide chain of 2,351 amino acids (aa) (SEQ ID No. 2) corresponding to a 19 aa signal peptide and a 2332 aa mature protein (330 kDa) (SEQ ID No. 3). The nucleotide sequence of the FVIII precursor is given in SEQ ID No. 1 and the corresponding protein sequence in SEQ ID No. 2. The FVIII precursor consists of a succession of the following seven functional domains: A1, a1, A2, a2, B, a3, A3, C1 and C2, from the N-terminal to the C-terminal (Vehar et al., 1984, Nature, 312:337-342).
FVIII undergoes a first intracellular proteolysis at arginines 1313 and 1648, producing a FVIII heterodimer consisting of: i) an A1-a1-A2-a2-B heavy chain; ii) an a3-A3-C1-C2 light chain. It circulates in plasma as a heterodimer. The interaction between the two chains is ensured among others by the presence of a chelated copper molecule in domains A1 and A3. Immediately after being secreted in plasma, FVIII forms a very high affinity association with von Willebrand factor (vWF) which protects it from proteases. FVIII and vWF form a noncovalent complex in which binding takes place mainly via two regions of FVIII: the N-terminal region and the C-terminal region at 2303-2332 (C2 domain) of the light chain. During coagulation, FVIII is cleaved by thrombin and factor Xa at three sites: i) thrombin cleaves at Arginine 1689 of the light chain and at Arginine 372 and Arginine 740 of the heavy chain; ii) factor XA cleaves FVIII at Arginine 336, Arginine 372 and Arginine 740. Two of these cleavages are common (Arginine 372 and Arginine 740). Cleavages at Arginine 372 and Arginine 1689 are essential for FVIII to participate in the coagulation cascade. These cleavages activate FVIII, also known as FVIIIa (“a” for “active”); in addition to FVIIIa activation, these cleavages result in removal of the 170 kDa B domain and dissociation of FVIIIa from vWF.
The B domain of FVIII, defined by amino acids 741 to 1648, can be totally or partially deleted with no loss of activity of recombinant FVIII (Toole et al., 1986, Proc. Natl. Acad. Sci. USA, 83 (16):5939-5942; Eaton et al., 1986, Biochemistry, 25 (26):8343-8347; Langer et al., 1988, Behring Inst. Mitt, 82:16-25; Meulien et al., 1988, Protein Eng, 2(4):301-6; and U.S. Pat. No. 4,868,112), including for porcine FVIII (U.S. Pat. No. 6,458,563; WO01/68109; U.S. Pat. No. 6,770,744), which in some cases can be used to replace the human FVIII.
Mutations, most of them point mutations, can be inserted at different sites of FVIII without causing a loss of FVIII procoagulant activity (U.S. Pat. Nos. 5,744,446; 5,859,204; 6,060,447; 6,180,371; 6,228,620; 6,376,463; EP 1561757; WO02/24723; WO97/49725). EP1502921 and WO2005/111074 describe human FVIIII variants with improved stability.
Other patents (US 2003/0083257; WO2005/040213; and U.S. Pat. No. 6,780,614) may be cited which describe modifications of FVIII cDNA for increasing its production in animal cells. The modifications of the cDNA are disclosed in patents US20021165177; US2002/0182684; EP1048726; EP1283263.
The number of units of FVIII administered is expressed in International Units (IU) with reference to the WHO standard for FVIII. FVIII activity is expressed either as a percentage (relative to normal human plasma) or in International Units (relative to an international standard). One International Unit (IU) of FVIII activity is equivalent to that quantity of FVIII contained in one milliliter of normal human plasma. Plasmatic FVIII assays may be carried out either by a chronometric method or by a chromogenic method.
Hemophilia A (severe and moderate forms) is generally treated by preventive or curative replacement therapy, which is based on repeated injections of the deficient coagulation factor or perfusion thereof. Patients with hemophilia A are treated with different types of plasma-derived or recombinant FVIII: i) recombinant; ii) semipurified plasma products; iii) plasma products purified on conventional or immunoaffinity columns. The first recombinant FVIII concentrates contained albumin as stabilizing agent. These included Kogenate® (Bayer), Helixate® (manufactured by Bayer, distributed by Aventis), and Recombinate® (Baxter). New albumin-free formulations have been developed, such as Kogenate® FS (Bayer), Helixate® FS (Bayer), and ReFactoMC (Wyeth). These nonetheless contain trace amounts of albumin arising from the cell culture medium used during the step of production of these recombinant proteins.
Recombinant human FVIII still needs to be optimized. Indeed, FVIII is relatively unstable in physiologic conditions, has a low activity in blood, is present at very low concentrations (0.1 to 0.2 μg/ml), and has a half-life of 10 to 12 hours.
In about 30% of severe hemophiliac A patients, replacement therapy causes complications specific to FVIII which lead to failure of the treatments usually used. In fact, after replacement therapy, patients may develop antibodies directed against the exogenous recombinant FVIII. These anti-FVIII antibodies inhibit the procoagulant activity of FVIII, hence the name “inhibitory antibodies” or else “inhibitors”. Further FVIII perfusion are rendered ineffective by these antibodies, and result in an increase of inhibitory antibody amount through a phenomenon known as “anamnestic reaction”.
Rapidly, patients can no longer be treated with FVIII, in which case the inhibitor “titer” is determined. This titer is expressed in international Bethesda units (BU). One BU of inhibitors corresponds to inactivation of half of the amount of FVIII in 1 ml of normal plasma. A titer is “low” when less than 10 BU, and “high” when more than 10 BU.
When the inhibitor titer is relatively low, hemophiliac patients may be given the aforementioned FVIII concentrates such as Kogenate® FS, Helixate® FS, Recombinate®, and ReFactoMC, but this carries a significant risk of inducing a rise in inhibitor titers which must therefore be closely monitored.
One of the ways to control inhibitory antibodies is to induce immune tolerance through administration of large doses of FVIII according to “de Bonn” protocol. In some patients, the inhibitory antibody titer is so high that they cannot be treated with large doses of FVIII for toxicity reasons.
A second approach known as the “Bonn-Malmo protocol” is based on one hand on ex vivo immunoadsorption of inhibitors immediately followed by reinjection of the blood, and on the other hand on injection of large doses of FVIII combined with immunosuppressive agents. These treatments are extremely costly in terms of recombinant FVIII and have achieved partial success.
Another approach consists in supplying coagulation factors in order to “bypass” the requirement of FVIII in the intrinsic coagulation pathway by using: i) plasma-derived activated prothrombin complex (FEIBA® VH, Factor Eight Inhibitor Bypassing Activity; Baxter) containing Factors II, VII, IX and X; ii) recombinant activated Factor VIIa (rFVIIa; NovoSeven®/Niastase®; NovoNordisk).
Said approaches have clear-cut success, nevertheless counterbalance by the development of side effects associated with this type of therapy (such as additional bleeding or conversely thrombotic events related to the frequency of administration).
It should be noted that circulating FVIII level increases after injection and then gradually declines related to its half-life. FVIII half-life ranges from 8 to 16 hours, with an average of 12 hours, raising the problem of repeated injections.
Another option consists in using a porcine FVIII with the aim to avoid antibodies directed against human FVIII. Patients who developed inhibitors to human FVIII have been successfully treated with semi-purified porcine FVIII (Hyate:C). Yet, this success has only been partial because after several injections of porcine FVIII, anti-porcine FVIII inhibitors have also developed, as mentioned in US2004/0249134. This phenomenon may necessitate to end treatment. Ipsen and Octagen are now co-developing a recombinant porcine FVIII known as OBI-1 in collaboration with Emory University in the USA, as a replacement for Hyate:C (WO2005107776).
Administration of porcine FVIII is therefore not a definitive solution for the treatment of hemophilia A patients with inhibitors.
As it can be seen, today there is no ideal treatment for individuals with hemophilia A, with or without inhibitors. The various problems encountered with commercial FVIII-based treatments associated with the development of these inhibitory antibodies have driven efforts to rapidly design a novel FVIII which has retained procoagulant specific activity and having lost the epitopes recognized by the inhibitory antibodies.
Few studies have addressed the epitope specificities of “inhibitory” antibodies. Some inhibitory antibodies appear to recognize small regions of the FVIII molecule: i) C2 domain in the light chain (2181-2321); ii) A2 domain in the heavy chain (484-509); iii) A3 domain (1694-2019) (Prescott et al., 1997, Blood, 89:3663-3671; Barrow et al., 2000, Blood, 95:557-561).
The 18 kDa C2 domain, between Serine 2173 and Tyrosine 2332, contains the membrane phospholipid binding domain and a part of the vWF binding domain. Inhibitory antibodies directed against the C2 domain mainly block the binding to phospholipids binding required for procoagulant activity but also the interaction with vWF. Mutations at positions Methionine 2199, Phenylalanine 2200, Valine 2223, Lysine 2227, Leucine 2251 and Leucine 2252 illustrate the importance of these amino acids in FVIIII activity and binding to phospholipids and/or to vWF (Pratt et al., 1999, Nature, 402:439-442).
Anti-A2 antibodies inhibit the function of FVIIIa as cofactor of Factor X (Lollar et al., 1994, J. Clin. Invest. 93:2497-2504). The main A2 epitope has been located between Arginine 484 and Leucine 508 (Healey et al., 1995, J. Biol. Chem., 270:14505-14509).
Antibodies directed against A3 and/or C2 domain prevent stabilization of the interaction between FVIII and vWF and also interfere with binding of the FVIII light chain to activated FIX.
Inhibitors are very heterogeneous from one patient to another and epitope specificity may change over time. Kinetic study of FVIII inhibition have revealed two types of allo-antibodies: type I antibodies which completely neutralize exogenous FVIII, and type II antibodies which never totally inhibit FVIII activity. Type II antibodies not completely block the procoagulant activity of FVIII because they are not saturable or display decreasing affinity according to their concentration.
Regions which can be recognized by inhibitory antibodies are cited in patents US2003/147900 and WO00/48635. These exposed and antigenic FVIII regions are between positions 1649-2019, 108-355, 403-725 and 2085-2249.
Moreover, US 2005/0256304 describes the following set of positions in human FVIII, where substitutions are likely to decrease antigenicity: 197, 198, 199, 201, 202, 407, 411, 412, 419, 515, 517, 613, 617, 636, 637, 638, 639, 823, 1011, 1013, 1208, 1209, 1210, 1254, 1255, 1257, 1262, 1264, 1268, 1119, 1120, 1121, 1122, and 1123.
The antigenicity of human FVIII can be decreased by glycosylation of recognition sites of inhibitors. Said method is disclosed in U.S. Pat. No. 6,759,216 and JP2004141173.
Another option consists in substituting the human FVIII epitopes usually recognized by inhibitors in domains: i) A2 (484-509); ii) A3 (1694-2019), a3 (1649-1687); iii) C2 (2181-2321). This solution is based on the use of a hybrid recombinant protein: a human/porcine FVIII.
The main targets of inhibitory antibodies are located in the A2 and C2 domains of FVIII (Saenko et al., Haemophilia, 2002). In fact, it is generally thought that 90% of inhibitory antibodies are directed against the human A2 and C2 domains (Barrow et al., 2000, Blood, 95:564-569). Moreover, it has been shown that human inhibitors have weak activity against porcine FVIII (Koshihara et al., Blood, 1995).
It is therefore expected that a substitution of human FVIII epitopes by porcine sequences would lead to a hybrid molecule less reactive towards inhibitory antibodies. Thus, the human A2 and C2 domains were replaced by their corresponding porcine domains (Lubin et al., 1994, J. Biol. Chem., 269:8639-8641). However, once again, anti-porcine FVIII antibodies eventually developed during the treatment of patients with inhibitors.
Many patents describe human/animal FVIII hybrids having retained a procoagulant activity. Human/animal hybrid, as used herein, denotes any combination (substitution) of at least one amino acid between a human FVIII sequence and a FVIII sequence of animal origin. Said hybrids have been produced, on the one hand, by substituting regions (functional subunits or structural domains) by the corresponding animal regions. For instance, U.S. Pat. Nos. 5,888,974; 5,663,060; 5,583,209; EP1359222; U.S. Pat. No. 5,744,446; WO93/20093; and WO95/24427 provide hybrid FVIII molecules derived from combinations of heavy and light chains of human and non-human FVIII, and/or derived from combinations of human/porcine FVIII domains.
U.S. Pat. No. 5,744,446 describes human/porcine FVIII variants wherein sequences of the human A2 domain are substituted by the corresponding murine or porcine sequences. The substituted fragments of the A2 domain are: 373-540; 373-508, 445-508, 484-508, 404-508, 489-508 and 484-489.
U.S. Pat. No. 5,364,771 provides a method for purifying FVIII hybrids derived from combinations of light and heavy chains from human and non-human FVIII: human FVIII in which the A2 domain is replaced by the porcine A2 domain.
On the other hand, in some patents, said hybrids are formed by point substitutions of one or several amino acids of human FVIII by the corresponding amino acid(s) of animal origin (porcine, canine or murine). For example, US2004/0197875 discloses modifications in codon charges at certain positions of human FVIII. Said positions are defined related to porcine FVIII sequence. EP1454916 describes the introduction of porcine codons into the human cDNA.
Among these patents, studies have been addressed to develop human/porcine FVIII hybrids in the region of the A2 domain. EP1359222 describes a study of the porcine A2 domain sequence, with a view to generating such hybrid. US2003/166536; U.S. Pat. No. 6,376,463; WO00/71141 describe amino acid substitutions in human FVIII at key epitopes in the A2 domain, between positions 484 and 508: 486, 490, 491, 493, 494, 496, 498, 499, 500, 502, 503, 504, 505, 506, 507 for WO00/71141; and 485, 487, 488, 489, 492, 495, 501, 508 for U.S. Pat. No. 5,859,204. In particular, Alanine substitutions were made at positions: Arginine 484, Proline 485, Tyrosine 487, Serine 488, Arginine 489, Proline 492, Valine 495, Phenylalanine 501, and Isoleucine 508. These substitutions conferring decreased antigenicity might be of interest from a therapeutic standpoint.
Likewise, in U.S. Pat. No. 6,180,371, Arginine 484 is substituted by Serine, Proline 485 by Alanine, Arginine 489 by Glycine, Proline 492 by Leucine. With these variants, inhibition of the procoagulant function of FVIII by antibodies was alleviated or disappeared altogether. The therapeutic interest of a double or triple mutant at Arginine 484, Arginine 489 and Phenylalanine 501, where each codon is substituted with an Alanine, is suggested.
There are also patents disclosing FVIII variants in which the substitutions only affect the C2 domain.
US2004/249134; WO03/047507; WO02/24723; U.S. Pat. No. 6,770,744 describe substitutions at positions Methionine 2199, Phenylalanine 2200, Valine 2223, Lysine 2227, Leucine 2251 and Leucine 2252. Said substitutions were introduced into a FVIII lacking the B domain. Amino acids at positions 2215, 2220, 2320, 2195, 2196, 2290 and 2313 were substituted with an Alanine.
With regard to position 2223, Valine is replaced by an Alanine, by comparison between human and porcine FVIII. This mutation is mentioned in Pratt's article “Structure of the C2 domain of human FVIII” (Nature, 1999, 402:439-442) and in U.S. Pat. No. 6,770,744.
Combinations of certain mutated positions such as 2199, 2200, 2223 and 2227 have been described as reducing the antigenicity of FVIII with regard to some anti-C2 domain inhibitory antibodies, all while retaining the coagulant activity of FVIII.
In patents WO99/46274 and US2005/0079584, J. Lollar's group describes a region of potential interest for constituting a less immunogenic FVIII: 2181 to 2243. This region was defined very roughly by an antigenicity study of human/porcine hybrids. An alignment between human and porcine FVIII of the sequence 2181 to 2243 disclosed 17 differences at the following positions: 2181, 2182, 2195, 2196, 2197, 2199, 2207, 2216, 2222, 2224, 2225, 2226, 2227, 2228, 2234, 2238, 2243. J. Lollar's group speculate that a substitution at these 17 positions by an Alanine, a Methionine, a Serine, a Glycine, or else a Leucine might generate a FVIII protein that can avoid inhibitory antibodies. This hypothesis is not supported by any antigenicity studies of mutants of interest.
Lastly, patents such as U.S. Pat. No. 6,180,371; US2002/182670; US 2003/068785; US2005/079584; WO99/46274; U.S. Pat. No. 7,012,132; WO2005/046583 provide human/porcine hybrids harboring substitutions in both the A2 and C2 domains of FVIII with the aim of reducing inhibition by inhibitory antibodies that recognize both domains. In particular, WO2005/046583 describes amino acid substitutions in the A2 and C2 domains at positions 484, 489, 492, 2199, 2200, 2251 and 2252. The FVIII which was used lacks the B domain. Only position 484 has an Arginine substituted by an Alanine.
To summarize, while many studies make reference to novel FVIII variants, there is still a need for a novel, less immunogenic FVIII, because there are no modified FVIII variants capable of treating patients with inhibitors currently on the market. Moreover, variants with an improved specific activity or an improved capacity to be secreted are also of major interest to promote the production of recombinant FVIII or to improve the treatment of patients.